Involvement of BCRP (ABCG2)in the Biliary Excretion of Pitavastatin
Masaru Hirano,Kazuya Maeda,Soichiro Matsushima,Yoshitane Nozaki,Hiroyuki Kusuhara,and Yuichi Sugiyama
Graduate School of Pharmaceutical Sciences,The University of Tokyo,Tokyo,Japan
Received April 20,2005;accepted June 13,2005
ABSTRACT
Pitavastatin,a novel potent 3-hydroxymethylglutaryl coenzyme A reductase inhibitor,is distributed selectively to the liver and excreted into bile in unchanged form in rats.We reported previously that the hepatic uptake is mainly mediated by or-ganic anion transporting polypeptide (OATP)1B1,whereas the biliary excretion mechanism remains to be clarified.In the present study,we investigated the role of breast cancer resis-tance protein (BCRP)in the biliary excretion of pitavastatin.The ATP-dependent uptake of pitavastatin by human and mouse BCRP-expressing membrane vesicles was significantly higher compared with that by control vesicles with K m values of 5.73and 4.77?M,respectively.The biliary excretion clearance of pitavastatin in Bcrp1(?/?)mice was decreased to one-tenth of that in control mice.The biliary excretion of pitavastatin was unchanged between control and Eisai hyperbilirubinemic rats,
indicating a minor contribution of multidrug resistance-associ-ated protein (Mrp)2.This observation differs radically from that for a more hydrophilic statin,pravastatin,of which biliary ex-cretion is largely mediated by Mrp2.These data suggest that the biliary clearance of pitavastatin can be largely accounted for by BCRP in mice.In the case of humans,transcellular transport of pitavastatin was determined in the Madin-Darby canine kid-ney II cells expressing OATP1B1and human canalicular efflux transporters.A significant basal-to-apical transport of pitavas-tatin was observed in OATP1B1/MDR1and OATP1B1/MRP2double transfectants as well as OATP1B1/BCRP double trans-fectants,implying the involvement of multiple transporters in the biliary excretion of pitavastatin in humans.This is in con-trast to a previous belief that the biliary excretion of statins is mediated mainly by MRP2.
The liver plays an important role in the excretion of xeno-biotics,including many kinds of drugs.A number of reports have shown that several kinds of transporters are expressed on the canalicular membrane in the liver for the efficient elimination of drugs via the bile (Chandra and Brouwer,2004).It has been generally accepted that transport of vari-ous organic anions across the canalicular membrane is mainly mediated by multidrug resistance-associated protein 2(MRP2/ABCC2),whereas the bile salt export pump (BSEP/ABCB11)exclusively accepts bile acids,and multidrug resis-tance protein (P-glycoprotein,MDR1/ABCB1)can transport relatively hydrophobic neutral or cationic compounds (Chan-dra and Brouwer,2004).However,several reports have
shown that some anionic drugs can also be recognized by BSEP and MDR1(Cvetkovic et al.,1999;Hirano et al.,2005),suggesting that multiple transport mechanisms are involved in the biliary excretion of organic anions.
Moreover,breast cancer resistance protein (BCRP/ABCG2)has been cloned recently and can accept various kinds of organic anions,especially sulfated conjugates of steroids and xenobiotics (Allikmets et al.,1998;Suzuki et al.,2003).Be-cause BCRP is expressed on the bile canalicular membrane of hepatocytes as well as the brush-border membrane of entero-cytes,trophoblast cells in placenta,and the apical membrane of lactiferous ducts in the mammary gland (Maliepaard et al.,2001),BCRP must also be considered as one of the routes for the biliary excretion of organic anions.Current evidence indicates that BCRP contributes to the membrane transport of some substrates,such as intestinal absorption and trans-fer to breast milk (Jonker et al.,2000,2002;Adachi et al.,2004;Mizuno et al.,2004;Kondo et al.,2005;Merino et al.,
This work was supported by a Health and Labor Sciences Research Grants from Ministry of Health,Labor,and Welfare for the Research on Advanced Medical Technology.
Article,publication date,and citation information can be found at https://www.doczj.com/doc/f87061616.html,.doi:10.1124/mol.105.014019.ABBREVIATIONS:MRP (Mrp),multidrug resistance-associated protein;OATP (Oatp),organic anion transporting polypeptide;BCRP (Bcrp),breast cancer resistance protein;BSEP (Bsep),bile salt export pump;MDR (Mdr),multidrug resistance protein;HEK,human embryonic kidney;MDCK,Madin-Darby canine kidney;HMG-CoA,3-hydroxy-3-methylglutaryl-coenzyme A;E 17?G,17?-estradiol-17?--glucuronide;EHBR,Eisai 0026-895X/05/6803-800–807$20.00M OLECULAR P HARMACOLOGY
Vol.68,No.3Copyright ?2005The American Society for Pharmacology and Experimental Therapeutics 14019/3048547Mol Pharmacol 68:800–807,2005
Printed in U.S.A.
at Central South Univ on June 18, 2014
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2005a).Regarding the involvement of BCRP in biliary excre-tion,some reports have demonstrated that the biliary excre-tion of2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine and nitrofurantoin is almost impaired in Bcrp1(?/?)mice (van Herwaarden et al.,2003;Merino et al.,2005a)and that the biliary excretion of topotecan and cimetidine is also mainly regulated by Bcrp,considering the gender difference in the hepatic expression level of Bcrp and the plasma con-centration profiles(Merino et al.,2005b).In addition,it has been demonstrated that certain kinds of single-nucleotide polymorphisms(SNPs)in BCRP and inhibition of BCRP-mediated transport by some compounds may alter its func-tion and the pharmacokinetics of some drugs in vitro(Imai et al.,2002;Zamber et al.,2003;Kondo et al.,2004)as well as in vivo(Kruijtzer et al.,2002;Sparreboom et al.,2004). Therefore,the pharmacokinetics of several important com-pounds are regulated mainly by BCRP.
Pitavastatin is a highly potent inhibitor of3-hydroxymeth-ylglutaryl coenzyme A(HMG-CoA)reductase,the rate-limit-ing enzyme in cholesterol biosynthesis(Kajinami et al., 2003).Pitavastatin causes a significant reduction in not only serum total cholesterol and low-density lipoprotein choles-terol but also triglyceride levels(Stein et al.,1998).It has been demonstrated that[14C]pitavastatin is distributed se-lectively to the liver in rats with a liver-to-plasma concentra-tion ratio of more than50(Kimata et al.,1998).We demon-strated previously that pitavastatin is taken up into human hepatocytes mainly by OATP1B1(OATP2/OATP-C)(Hirano et al.,2004).Then,because it is scarcely metabolized in human liver microsomes(Fujino et al.,2003),pitavastatin is supposed to be excreted into bile in unchanged form(Kojima et al.,2001).However,the biliary transport mechanism of pitavastatin has not been clarified yet.So far,Fujino et al. (2002)have demonstrated that after intravenous bolus ad-ministration of pitavastatin,its plasma concentration and biliary excretion in Eisai hyperbilirubinemic rats(EHBRs), an Mrp2-deficient rat,are comparable with those in Sprague-Dawley rats and that the plasma and brain concentrations of pitavastatin in mdr1a/1b knockout mice are not different from those in control mice.In addition,we have already shown that pitavastatin is not a substrate of rat and human BSEP(Hirano et al.,2005).These results suggest that BSEP, MRP2,and MDR1do not make a major contribution to the biliary excretion of pitavastatin.
In the present study,to demonstrate the involvement of BCRP in the biliary excretion of pitavastatin,we examined the transport of pitavastatin by transporter expression sys-tems and observed the biliary excretion clearance of pitavas-tatin in transporter-deficient animals.Moreover,to check whether other statins could be substrates of BCRP or not,we performed a transport study using BCRP-expressing mem-brane vesicles.
Materials and Methods
Animals.Male Bcrp1(?/?)and wild-type FVB mice(16–18weeks old)were used in the present study(Jonker et al.,2002).Male Sprague-Dawley rats(7–8weeks old)and EHBRs(7–8weeks old) were purchased from Nippon SLC(Shizuoka,Japan).All animals were maintained under standard conditions with a reverse dark-light cycle and were treated humanely.Food and water were avail-Animal Care Committee(Graduate School of Pharmaceutical Sci-
ences,The University of Tokyo,Tokyo,Japan).
Materials.Pitavastatin,monocalcium bis[(3R,5S,6E)-7-[2-cyclo-propyl-4-(4-fluorophenyl)-3-quinolyl]3,5-dihydroxy-6-hepteonate] was synthesized by Nissan Chemical Industries(Chiba,Japan). [3H]Pitavastatin(16.0Ci/mmol)and[3H]cerivastatin(4.86Ci/mmol) were synthesized by Amersham Biosciences UK,Ltd.(Little Chal-font,Buckinghamshire,UK)and Hartmann Analytic GmbH(Braun-schweig,Germany),respectively.[3H]Pravastatin(44.6Ci/mmol), [14C]fluvastatin(45.7mCi/mmol),and[3H]rosuvastatin(79Ci/mmol) were supplied by Sankyo Co.,Ltd.(Tokyo,Japan),Novartis Pharma K.K.(Basel,Switzerland),and AstraZeneca PLC(London,UK),re-spectively.Other unlabeled HMG-CoA reductase inhibitors(atorva-statin,cerivastatin,fluvastatin,pravastatin,rosuvastatin,and sim-vastatin acid)were donated by Kowa Co.,Ltd.(Tokyo,Japan). [3H]17?-Estradiol-17?-D-glucuronide(E
2
17?G)and[3H]estrone-3-sulfate(45and46Ci/mmol,respectively)were purchased from PerkinElmer Life and Analytical Sciences(Boston,MA).Unlabeled
E
2
17?G and estrone-3-sulfate were purchased from Sigma-Aldrich (St.Louis,MO).All other chemicals were of analytical grade and were commercially available.
Cell Culture.Human BCRP-,mouse Bcrp-,or green fluorescent protein(GFP)-transfected HEK293cells(Kondo et al.,2004)and MDCK II single-and double-transfected cells expressing human OATP1B1,MDR1,MRP2,or BCRP(Matsushima et al.,2005)and rat Oatp1b2or Mrp2(Sasaki et al.,2004)were grown in Dulbecco’s modified Eagle’s medium low glucose(Invitrogen,Carlsbad,CA) supplemented with10%fetal bovine serum(Sigma-Aldrich),100
U/ml penicillin,and100?g/ml streptomycin at37°C with5%CO
2 and at95%humidity.
Construction and Infection of Recombinant Adenovirus and the Membrane-Vesicle Preparation.Details of the proce-dure for producing recombinant adenovirus containing human BCRP,mouse Bcrp,and GFP have been described previously(Kondo et al.,2004).Membrane vesicles were prepared from BCRP and GFP-transfected HEK293cells according to the method described previously(Kondo et al.,2004).For the preparation of the isolated membrane vesicles,HEK293cells cultured in a15-cm dish were infected by recombinant adenovirus containing human and mouse BCRP cDNA(multiplicity of infection?10).As a negative control, cells were infected with GFP(multiplicity of infection?10).Cells were harvested48h after infection,and then the membrane vesicles were isolated from1to2?108cells using a standard method described previously in detail(Muller et al.,1994).In brief,cells were diluted40-fold with hypotonic buffer(1mM Tris-HCl and0.1mM EDTA,pH7.4,at37°C)and stirred gently for1h on ice in the presence of2mM phenylmethylsulfonyl fluoride,5?g/ml leupeptin, 1?g/ml pepstatin,and5?g/ml aprotinin.The cell lysate was cen-trifuged at100,000g for30min at4°C,and the resulting pellet was suspended in10ml of isotonic TS buffer(10mM Tris-HCl and250 mM sucrose,pH7.4at4°C)and homogenized using a Dounce B homogenizer(glass/glass,tight pestle,30strokes).The crude mem-brane fraction was layered on top of38%(w/v)sucrose solution in5 mM Tris-HEPES,pH7.4,at4°C,and centrifuged in Beckman SW41 rotor centrifuged at280,000g for60min at4°C.The turbid layer at the interface was collected,diluted to23ml with TS buffer,and centrifuged at100,000g for30min at4°C.The resulting pellet was suspended in400?l of TS buffer.Vesicles were formed by passing the suspension30times through a27-gauge needle using a syringe. The membrane vesicles were finally frozen in liquid nitrogen and stored at?80°C until use.Protein concentrations were determined by the Lowry method,and bovine serum albumin was used as a standard.
Transport Studies with Membrane Vesicles.The transport studies were performed using a rapid filtration technique(Hirohashi et al.,1999).In brief,15?l of transport medium(10mM Tris-HCl, BCRP-Mediated Efflux Transport of Pitavastatin801
at37°C for3min and then rapidly mixed with5?l of membrane vesicle suspension(5?g of protein).The reaction mixture contained 5mM ATP or AMP along with the ATP-regenerating system(10mM creatine phosphate and100?g/?l creatine phosphokinase).The transport reaction was terminated by the addition of1ml of ice-cold buffer containing10mM Tris-HCl,250mM sucrose,and0.1M NaCl, pH7.4.The stopped reaction mixture was filtered through a0.45-?m hemagglutinin filter(Millipore Corporation,Billerica,MA)and then washed twice with5ml of stop solution.Radioactivity retained on the filter was determined in a liquid scintillation counter (LS6000SE;Beckman Coulter,Inc.,Fullerton,CA)after the addition of scintillation cocktail(Clear-sol I;Nacalai Tesque,Tokyo,Japan).
Transcellular Transport Study.Transfected MDCK II cells were seeded in24-well plates at a density of1.4?105cells/well and were cultured with10mM sodium butyrate for24h before the transport study(Sasaki et al.,2002).Krebs-Henseleit buffer con-
sisted of142mM NaCl,23.8mM Na
2CO
3
,4.83mM KCl,0.96mM
KH
2PO
4
,1.20mM MgSO
4
,12.5mM HEPES,5mM glucose,and1.53
mM CaCl
2
adjusted to pH7.4.The experiments were initiated by
replacing the medium on either the apical or basal side of the cell layer with complete medium containing tritium-labeled and unla-
beled E
2
17?G or pitavastatin(0.1?M).The cells were incubated at 37°C,and aliquots of medium were taken from each compartment at designated time points.Radioactivity in100?l of medium was mea-sured in a liquid scintillation counter(LS6000SE;Beckman Coulter) after the addition of scintillation cocktail(Clear-sol I;Nacalai Tesque).At the end of the experiments,the cells were washed three times with1.5ml of ice-cold Krebs-Henseleit buffer and solubilized in450?l of0.2N NaOH.After the addition of225?l of0.4N HCl, 600-?l aliquots were transferred to scintillation vials.Aliquots(50?l)of cell lysate were used to determine protein concentrations as described above.
Kinetic Analysis.Ligand uptake was normalized in terms of the amount of membrane protein and expressed as the uptake volume [?l/mg protein],given as the amount of radioactivity associated with the cells[dpm/mg protein]divided by its concentration in the incu-bation medium[dpm/?l].The ATP-dependent uptake of ligands via BCRP was calculated by subtracting the ligand in the presence of AMP from that in the presence of ATP.Kinetic parameters were
obtained using the equation v?(V
max?S)/(K m?S),where v is the uptake velocity of the substrate(in picomoles per minute per milli-gram of protein),S is the substrate concentration in the medium(in micromolars),K
m
is the Michaelis constant(in micromolars),and
V
max
is the maximum uptake velocity(in picomoles per minute per milligram of protein).The Damping Gauss Newton Method algo-rithm was used with a MULTI program(Yamaoka et al.,1981)to perform nonlinear least-squares data fitting.Inhibition constants
(K
i
)of a series of compounds were calculated with the use of the following equation(if the substrate concentration was much lower
than the K
m value):CL(?I)?CL/(1?I/K
i
),where CL represents the
uptake clearance in the absence of inhibitor,CL(?I)represents the uptake clearance in the presence of inhibitor,and I represents the concentration of the inhibitor.When fitting the data to determine the K
i
value,the input data were weighed as the reciprocal of the observed values.
In Vivo Infusion Study in Rats.Male Sprague-Dawley rats and EHBRs weighing approximately250to300g were used for these experiments.Under pentobarbital anesthesia(30mg/kg),the femo-ral vein was cannulated with a polyethylene catheter(PE-50)for the injection of[3H]pitavastatin.The bile duct was cannulated with a polyethylene catheter(PE-10)for bile collection.The rats received a constant infusion of pitavastatin at a dose of72?g/h/kg after a bolus intravenous administration of0.25mg/kg.Blood samples were col-lected from the jugular vein.Bile was collected in preweighed test tubes at30-min intervals throughout the experiment.Plasma was prepared by centrifugation of the blood samples(10,000g,Microfuge;minced,and subsequently200?l of hydroxyperoxide and400?l of
isopropanol were added to approximately200mg of liver.This was incubated at55°C for4to6h after the addition of2ml of soluene (PerkinElmer Life and Analytical Sciences)to dissolve the tissues, and then the radioactivity was determined in a liquid scintillation counter after the addition of scintillation cocktail.
In Vivo Infusion Study in Mice and Quantification of Pitavastatin by LC/MS.Male FVB and Bcrp1(?/?)mice weighing approximately28to33g were used throughout these experiments. Under pentobarbital anesthesia(30mg/kg),the jugular vein was cannulated with a polyethylene catheter(PE-10)for the injection of pitavastatin.The bile duct was cannulated with a polyethylene cath-eter(SP-8)for bile collection.The mice received a constant infusion of pitavastatin at a dose of300?g/h/kg.Blood samples were collected from the opposite jugular vein.Bile was collected in preweighed test tubes at15-min intervals throughout the experiments.Plasma was prepared by centrifugation of the blood samples.The mice were killed after150min,and the entire liver,kidney,brain,and skeletal muscle were excised immediately.The tissues were weighed and stored at?80°C until quantification.Portions of liver,kidney,brain, and skeletal muscle were added to five volumes of physiological saline(w/v)and homogenized.Plasma(10?l),bile(1?l),or tissue homogenate(10or100?l)was deproteinized with200?l of methanol containing the internal standard(40ng/ml atorvastatin),followed by centrifugation at4°C and10,000g for10min.The supernatant(100?l)was mixed with50?l of water and subjected to high-performance liquid chromatography(Waters2695;Waters,Milford,MA).The LC/MS analysis of pitavastatin was performed using an Inertsil ODS-3column(50?2.1mm;particle size,5?m)(GL Sciences, Tokyo,Japan).The mobile phase consisted of methanol/ammonium formate buffer,pH4?7:3(v/v),and the flow rate was0.7ml/min. The MS instrument used for this work was a ZQ micromass(Waters) equipped with a Z-spray source;it was operated in the positive-ion electrospray ionization mode.The Z-spray desolvation temperature, capillary voltage,and cone voltage were350°C,3400V,and40V, respectively.The m/z monitored for pitavastatin and atorvastatin was422.3and559.0,respectively.No chromatographic interference was found for pitavastatin and atorvastatin in extracts from blank plasma,bile,and tissue homogenates.The retention times of pitavastatin and atorvastatin were1.2and1.1min,respectively.The detection limits for pitavastatin were5,2000,100,5,5,and5ng/ml in plasma,bile,liver,kidney,brain,and muscle,respectively.
Pharmacokinetic Analysis.Total plasma clearance(CL
total
),
biliary clearance normalized by circulating plasma(CL
bile,plasma
), and biliary clearance normalized by the liver concentration
(CL
bile,liver
)were calculated from the equations CL
total?I/C ss,plasma, CL
bile,plasma?V bile/C ss,plasma,and CL bile,liver?V bile/C ss,liver,where I,C
ss,plasma
,V
bile
,and C
ss,liver
represent the infusion rate(in micro-grams per minute per kilogram),plasma concentrations at steady state(in nanograms per milliliter),biliary excretion rate at steady state(in micrograms per minute per kilogram),and hepatic concen-tration at steady state(in nanograms per milliliter),respectively.In
rats,C
ss,plasma
was determined as the mean value of the plasma [3H]pitavastatin concentrations at60,90,120,150,180,and210
min.V
bile
was determined as the mean value of the biliary excretion rate of[3H]pitavastatin from60to90min,from90to120min,from 120to150min,from150to180min,and from180to210min.In
mice,C
ss,plasma
was determined as the mean value of the plasma
unlabeled pitavastatin concentrations at90,120,and150min.V
bile was determined as the mean value of the biliary excretion rate of unlabeled pitavastatin from90to105min,from105to120min,from 120to135min,and from135to150min.C
ss,liver
was determined as the hepatic pitavastatin concentration at the end of the in vivo
experiment.To calculate C
ss
,
liver
,the specific gravity of the liver was assumed to be unity.Thus,the amount in the liver(nanograms per gram of liver)can be regarded as the hepatic concentration(nano-
802Hirano et al.
Statistical Analysis.Statistically significant differences were determined using one-way analysis of variance followed by Fisher’s least significant difference method.Differences were considered to be significant at P ?0.05.
Results
ATP-Dependent Uptake of [3H]Pitavastatin into BCRP-Expressing Membrane Vesicles.The time profiles and Eadie-Hofstee plots for the uptake of [3H]pitavastatin by BCRP-expressing membrane vesicles are shown in Fig.1.The uptake of [3H]pitavastatin into membrane vesicles from
human and mouse BCRP-transfected HEK293cells was markedly stimulated by ATP but not that into GFP-trans-fected control cells (Fig.1,A and B).The concentration-dependence of human and mouse BCRP-mediated ATP-de-pendent uptake of pitavastatin is shown in Fig.1,C and D.The K m and V max values for the ATP-dependent uptake of pitavastatin were 5.73? 1.52?M and 1106?79pmol/min/mg protein by human BCRP and 4.77?0.50?M and 881?20pmol/min/mg protein by mouse Bcrp,respectively.Uptake of Other Statins into BCRP-Expressing Membrane Vesicles.Uptake of other statins into human and mouse BCRP-expressing membrane vesicles was ob-served (Fig.2).We did not see any significant ATP-depen-dent uptake of cerivastatin and fluvastatin by mouse Bcrp-expressing membrane vesicles compared with GFP-transfected vesicles,whereas human BCRP significantly rec-ognized all of the statins we tested (cerivastatin,fluvastatin,pitavastatin,pravastatin,and rosuvastatin)as a substrate (Fig.2,A–E).Estrone-3-sulfate (0.1?M),which was a posi-tive control compound for BCRP-mediated transport,was accepted as a substrate of both human and mouse BCRP (Fig.2F).The K m value of estrone-3-sulfate by mouse Bcrp was 16.4?3.0?M (data not shown).
Inhibitory Effects of Statins on the ATP-Dependent Uptake of [3H]Estrone-3-sulfate into Human and Mouse BCRP-Expressing Membrane Vesicles.The in-hibitory effects of statins on the ATP-dependent uptake of [3H]estrone-3-sulfate by human and mouse BCRP-express-ing membrane vesicles were observed.All of the statins except for pravastatin (?300?M),inhibited the ATP-de-pendent uptake of [3H]estrone-3-sulfate by human and mouse BCRP in a dose-dependent manner.The K i values of statins for human and mouse BCRP are summarized in Table 1.
Transcellular Transport of Pitavastatin across Dou-ble Transfectants.Transcellular transport of pitavastatin across double-transfected MDCK II monolayers expressing uptake transporter (OATP1B1)and efflux transporter (MDR1,MRP2,or BCRP)was compared with that across the single-transfected monolayer and the vector-transfected
con-
Fig.1.Time profiles and Eadie-Hofstee plots of the uptake of [3H]pitavas-tatin by human and mouse BCRP-expressing membrane vesicles.The uptake of 0.1?M [3H]pitavastatin by human BCRP (hBCRP)(A)and mouse Bcrp (mBcrp)(B),respectively,was examined at 37°C in medium containing 5mM ATP (closed symbols)or AMP (open symbols).Circles and triangles represent the uptake in hBCRP-(or mBcrp-)and GFP-expressing membrane vesicles,respectively.The hBCRP-(C)and mBcrp (D)-mediated uptake of [3H]pitavastatin was determined for 2and 1min,respectively.Each point represents the mean ?S.E.(n ?3).Where vertical bars are not shown,the S.E.values are within the limits of the symbols.The data were fitted to the Michaelis-Menten equation by non-linear regression analysis,as described under Materials and Methods ,and each solid line represents the fitted
curve.
Fig.2.The ATP-dependent uptake of statins by human BCRP (hBCRP)-,mouse Bcrp (mBcrp)-,and GFP-expressing membrane vesicles.The uptake of 0.1?M [3H]cerivasta-tin (A),5?M [14C]fluvastatin (B),0.1?M [3H]pitavastatin (C),0.1?M [3H]pravastatin (D),and 0.1?M [3H]rosuvastatin (E)for 5min and 0.1?M [3H]estrone-3-sulfate (F)for 2min,respectively,was determined at 37°C in medium in the presence of 5mM ATP (f )or AMP (Ⅺ).Each point represents the mean ?S.E.(n ?3).?,P ?0.05;??,P ?0.01
BCRP-Mediated Efflux Transport of Pitavastatin
803
trol monolayer.In the case of humans,as shown in Fig.3,a symmetrical flux of pitavastatin was observed across the control,MDR1-,MRP2-,and BCRP-expressing MDCK II monolayer.The basal-to-apical flux of pitavastatin across the OATP1B1-expressing monolayer was approximately twice that in the opposite direction,whereas the basal-to-apical flux of pitavastatin was approximately 15,110,and 230times higher than that in the opposite direction in OATP1B1/MRP2,OATP1B1/BCRP,and OATP1B1/MDR1double-trans-fected cells,respectively.In addition,transcellular transport of pitavastatin across double-transfected MDCK II monolay-ers expressing rat Oatp1b2and Mrp2was also compared with that across the Oatp1b2or Mrp2single-transfected monolayer and the vector-transfected control monolayer.The basal-to-apical flux of pitavastatin was 2.4times higher than that in the opposite direction in the Oatp1b2/Mrp2double-transfected cells,although a smaller vectorial transport was observed even in the Oatp1b2-expressing monolayer (Fig.4).In both rats and humans,we checked the transcellular trans-port of E 217?G as a positive control in parallel and obtained results similar to those measured previously (Matsushima et al.,2005;data not shown).
Plasma,Liver Concentration and Biliary Excretion Profiles at Steady State in Sprague-Dawley Rats and EHBRs.It has been reported that pitavastatin is not signif-icantly metabolized in rats,and 70%of the excreted amount in bile can be detected in unchanged form (Fujino et al.,2002).Therefore,we used [3H]pitavastatin in rats in an in vivo study to examine the involvement of Mrp2in the biliary excretion of pitavastatin.The plasma concentration of pitavastatin reached a plateau at 60min during constant infusion after a bolus intravenous administration.The bili-ary excretion rate (V bile ),the steady-state plasma concentra-tion (C ss,plasma ),and total clearance (CL total )values of pitavastatin in EHBRs were almost the same as those in normal rats (Fig.5and Table 2).The concentration of pitavastatin and the K p value in the liver at steady state in EHBRs were lower than those in normal rats,and the CL bile -,plasma and CL bile,liver values in EHBRs were slightly higher than those in normal rats,although the differences were not statistically significant (Fig.5).
Plasma,Tissue Concentration,and Biliary Excre-tion Profiles at Steady State in FVB and Bcrp1(?/?)Mice.The plasma concentration of pitavastatin reached a plateau at 90min during constant infusion (Fig.6).The V bile of parent pitavastatin in Bcrp1(?/?)mice was much lower than that in control mice,whereas the C ss,plasma and CL total values of pitavastatin in Bcrp1(?/?)mice did not differ from those in normal mice (Table 3).The K p values of pitavastatin in the kidney,brain,and skeletal muscle as well as the liver (Table 3)at steady state were similar in control and Bcrp1(?/?)mice (K p value for kidney,1.41?0.10and 1.48?0.12ng/g;for brain,0.0308?0.0027and 0.0322?0.0070ng/g;and for skeletal muscle,0.0985?0.0075and 0.0960?0.0061ng/g in wild-type and Bcrp1(?/?)mice,respectively).
Discussion
Pitavastatin,a new potent inhibitor of HMG-CoA reduc-tase,is scarcely metabolized in humans and is believed to be excreted into the bile in intact form.In the present study,we concentrated on a new candidate transporter,BCRP,for the biliary excretion of
pitavastatin.
Fig.3.Time profiles for the transcellular transport of [3H]pitavastatin across MDCK II monolayers expressing human transporters.Transcellular transport of [3H]pitavastatin (0.1?M)across MDCK II monolayers expressing OATP1B1(B),BCRP (C),MDR1(D),MRP2(E),both OATP1B1and BCRP (F),both OATP1B1and MDR1(G),and both OATP1B1and MRP2(H)was compared with that across the vector-transfected control MDCK II monolayer (A).E and F represent the transcellular transport in the apical-to-basal and basal-to-apical direction,re-spectively.Each point represents the mean ?S.E.(n ?3).Where vertical bars are not shown,the S.E.values are within the limits of the symbols.
TABLE 1
Inhibitory effects of statins on the ATP-dependent uptake of
?3H ?estrone-3-sulfate by human BCRP-and mouse Bcrp-expressing membrane vesicles
The ATP-dependent uptake of ?3H ?estrone-3-sulfate (0.1?M)for 1min by human BCRP (hBCRP)and mouse Bcrp (mBcrp)was determined in the presence and absence of unlabeled statins at the designated concentrations.The values are expressed as a percentage of the ATP-dependent uptake of ?3H ?estrone-3-sulfate in the absence of any unlabeled compounds.The ATP-dependent uptake was obtained by subtracting the transport velocity in the presence of AMP from that in the presence of ATP.The K i values were determined by fitting the data using the non-least squares method.The values are expressed as mean ?computer-calculated S.D.(n ?3).
Statins
hBCRP
mBcrp
?M
Atorvastatin 14.3?1.917.6?1.7Cerivastatin 18.1?2.530.1?6.3Fluvastatin 5.43?0.278.62?0.86Pitavastatin 2.92?0.35 6.00?0.71Pravastatin ?300?M ?300?M Rosuvastatin 15.4?2.410.3?0.6Simvastatin acid 18.0?4.324.9?3.8
804
Hirano et al.
ATP-dependent uptake of pitavastatin in human and mouse BCRP-expressing membrane vesicles was observed (Fig.1).The K m value of pitavastatin for human BCRP (5.73?M)was almost the same as that for mouse Bcrp (4.77?M),and these K m values were similar to that of estrone-3-sulfate,which is a well-known substrate of human BCRP (Suzuki et al.,2003),indicating that pitavastatin is also a good sub-strate of BCRP.Regarding other statins,as shown in Table 1,no species difference in the K i value of each statin for the BCRP-mediated uptake of estrone-3-sulfate was observed between humans and mice.The inhibition potency of statins,except for pravastatin,was almost identical,whereas prava-statin could not inhibit the BCRP-mediated transport of es-
trone-3-sulfate up to 300?M.On the other hand,all statins we tested could be substrates of human BCRP,whereas four of them were also substrates of mouse Bcrp (Fig.2).From a pharmacokinetic viewpoint,BCRP could at least partly con-tribute to the biliary excretion of pravastatin,pitavastatin,and rosuvastatin,which are scarcely metabolized by meta-bolic enzymes like cytochrome P450in the liver.On the other hand,in the case of atorvastatin,cerivastatin,and simvasta-tin,which have been reported to be extensively metabolized by cytochrome P450enzymes (Garcia et al.,2003),the biliary excretion of their unchanged form via BCRP was considered to be minor in in vivo situations.
To estimate the contribution of each transporter to the biliary excretion of pitavastatin in the in vivo study,the biliary excretion of pitavastatin at steady state was investi-gated using Mrp2-and Bcrp1-deficient animals.Because pitavastatin was found to be scarcely metabolized in rats (Fujino et al.,2002),the radioactivity would reflect the par-ent pitavastatin.We confirmed that the plasma concentra-tion and biliary excretion rate of pitavastatin was not changed between Sprague-Dawley rats and EHBRs (Fig.5and Table.2),which is consistent with a previous report (Fujino et al.,2002).Therefore,the involvement of Mrp2in the biliary excretion of pitavastatin is minor.The liver con-centration in EHBRs was significantly lower than that in control rats possibly because of the accelerated efflux via the basolateral membrane in the liver caused by the induction of Mrp3(Ogawa et al.,2000)and the inhibition of hepatic uptake process from the blood by a higher concentration of bilirubin glucuronide in EHBRs (Sathirakul et al.,1993),or by the decrease in the expression levels of transporters re-sponsible for the uptake of pitavastatin.
Biliary excretion of pitavastatin was also observed in Bcrp1(?/?)and control mice.In contrast to humans and rats,pitavastatin is extensively metabolized in mice,so we decided to quantify the unchanged form separately by LC/MS in mice.The biliary excretion clearance of pitavastatin in Bcrp1(?/?)mice was 10times lower than that in control mice,suggesting that unchanged pitavastatin is excreted into bile mainly by Bcrp.In control mice,the biliary
clear-
Fig.4.Time profiles for the transcellular transport of [3H]pitavastatin across MDCK II monolayers expressing rat transporters.Transcellular transport of [3H]pitavastatin (0.1?M)across MDCK II monolayers ex-pressing Oatp1b2(B),Mrp2(C),and both Oatp1b2and Mrp2(D)was compared with that across the vector-transfected control MDCK II mono-layer (A).E and F represent the transcellular transport in the apical-to-basal and basal-to-apical direction,respectively.Each point represents the mean ?S.E.(n ?3).Where vertical bars are not shown,the S.E.values are within the limits of the
symbols.
Fig.5.Plasma concentrations and biliary excretion rate of [3H]pitavastatin during constant intravenous infusion into Sprague-Dawley rats and EHBRs.The plasma concentra-tion (A)and biliary excretion rate (B)of total radioactivity were determined during constant intravenous infusion into Sprague-Dawley rats (E )and EHBRs (F ).Each point plot-ted,with its vertical bar,represents the mean ?S.E.of four rats.
TABLE 2
Pharmacokinetic parameters of pitavastatin during constant intravenous infusion into Sprague-Dawley (SD)rats and EHBRs
Data represent the mean ?S.E.(n ?4).
C ss,plasma CL total CL bile,plasma CL bile,liver Liver Concentration
K p,liver
ng ?Eq/ml
ml/min/kg
ml/min/kg
ml/min/kg
ng ?Eq/g
SD rat 195?13 6.26?0.49 4.55?0.130.248?0.0424346?40623.0?3.6EHBR 179?8 6.75?0.34 5.77?0.530.399?0.0492621?139**14.7?0.8
**P ?0.01.
BCRP-Mediated Efflux Transport of Pitavastatin
805
ance normalized by plasma concentration(CL
bile,plasma
)ac-
counted for only5%of the plasma total clearance(CL
total
), indicating that pitavastatin is mainly excreted into bile as metabolites.Taking extensive metabolism into consider-ation,it is natural that the plasma and liver concentrations were not different between Bcrp1(?/?)and control mice, even if the biliary clearance of the parent compound was drastically reduced in Bcrp1(?/?)mice.In contrast to mice, pitavastatin is believed to be excreted into the bile in an intact form with only minimal metabolism in humans(Fujino et al.,2003).Therefore,BCRP is estimated to be involved in the biliary excretion of pitavastatin in humans.On the other hand,a previous in vivo study using EHBRs revealed that Mrp2is a major transporter for the excretion of pravastatin in rats(Yamazaki et al.,1997).Even though pitavastatin and pravastatin belong to the same category,HMG-CoA reduc-tase inhibitors,it was interesting to find that pitavastatin and pravastatin are excreted into the bile by different efflux transport systems.In the brain and kidney,in which BCRP is also expressed,and skeletal muscle,which is a target site of severe adverse effects of statins(rhabdomyolysis),these tissue concentrations were not significantly different be-tween Bcrp1(?/?)and control mice.Thus,Bcrp does not contribute to the distribution of pitavastatin in these tissues. BCRP was expressed not only in the liver,kidney,and brain, but also in the small intestine and mammary gland(Doyle and Ross,2003).It has been demonstrated that BCRP criti-cally modulates the absorption of certain drugs from the small intestine and transfer to milk(Adachi et al.,2004; Merino et al.,2005a).Further studies are needed to clarify the importance of BCRP in the intestinal absorption and tissue distribution of a series of statins.
In Fig.3,we clearly observed the vectorial basal-to-apical transport of pitavastatin across all of the double transfec-tants(OATP1B1/BCRP,OATP1B1/MDR1,and OATP1B1/ MRP2)in contrast to that across the control and OATP1B1 single-transfected cells.The efflux transport clearance of pitavastatin normalized by the intracellular concentration did not differ much among MDR1,MRP2,and BCRP,the range being only2-fold.In the case of pravastatin,we dem-onstrated previously that the efflux clearance of MRP2was much higher than that of MDR1and BCRP(Matsushima et al.,2005).Considering these facts,the relative contribution of MDR1and BCRP to the biliary excretion of pitavastatin was larger than that of pravastatin.In the case of rats,the basal-to-apical flux of pitavastatin across the Oatp1b2/Mrp2 double transfectants was significantly higher than that across the Oatp1b2single transfectants(Fig.4).However, the ratio of basal-to-apical to apical-to-basal flux of pitavas-tatin in Oatp1b2/Mrp2double-transfected cells divided by that in Oatp1b2single-transfected cells was approximately 1.6,which was small in comparison with that of other sub-strates tested previously(Sasaki et al.,2004).Therefore, although pitavastatin is a substrate of rat Mrp2,the intrinsic efflux clearance of pitavastatin via rat Mrp2seems to be relatively low.
The elimination of diflomotecan from plasma has been found to be delayed in patients with frequently observed SNP in BCRP(C421A/Q141K)(Sparreboom et al.,2004).Various reports suggested that this SNP affected the protein expres-sion level of BCRP in the placenta without changing its intrinsic efflux clearance(Kondo et al.,2004;Kobayashi et al.,2005).On the other hand,both in vivo and in vitro studies revealed that the frequently observed haplotype OATP1B1*15(N130D and V174A)reduced the nonrenal clearance of pravastatin(Nishizato et al.,2003;Iwai et al., 2004).We demonstrated previously that pitavastatin is taken up into hepatocytes mainly by OATP1B1(Hirano et al., 2004).Taking these facts into consideration,further clinical studies are needed to investigate the effect of SNPs in OATP1B1and BCRP on the pharmacokinetics of pitavasta-tin and to determine the importance of each transporter directly in humans.
In the present study,we have shown that pitavastatin is recognized by human and mouse BCRP in expression sys-tems,and Bcrp is mainly involved in the biliary excretion of pitavastatin by knockout animals.BCRP can also accept other statins as a substrate,implying that it is important
to
Fig.6.Plasma concentration and biliary excretion rate of
pitavastatin during constant intravenous infusion into
wild-type and Bcrp1(?/?)mice.The plasma concentration
(A)and biliary excretion rate(B)of pitavastatin were de-
termined during constant intravenous infusion into wild-
type(E)and Bcrp1(?/?)(F)mice.Each point plotted,with
its vertical bar,represents the mean?S.E.of four mice.?,
P?0.05
TABLE3
Pharmacokinetic parameters of pitavastatin during constant intravenous infusion into wild-type and Bcrp1(?/?)mice
Data represent the mean?S.E.(n?4).
C ss,plasma CL total CL bile,plasma CL bile,liver Liver Concentration K p,liver
ng/ml ml/min/kg ml/min/kg ml/min/kg ng/g Wild type254?2120.1?1.8 1.21?0.430.364?0.032813?196 3.34?1.03 Bcrp1(?/?)213?2224.2?2.50.110?0.02*0.0293?0.0036**798?124 3.94?0.83
*P?0.05;**P?0.01.
806Hirano et al.
estimate how the change of function and expression level of BCRP affects the pharmacokinetics of statins.In humans, because it is evident that pitavastatin is a substrate of BCRP, MDR1,and MRP2by double transfectants,it will be impor-tant to estimate the contribution of each transporter to the biliary excretion of pitavastatin in humans.
Acknowledgments
We thank Dr.Alfred H Schinkel(The Netherlands Cancer Insti-tute,the Netherlands)and Dr.Johan A Jonker(The Salk Institute for Biological Studies,San Diego,CA)for constructing Bcrp1(?/?) mice and giving us valuable comments,Dr.Piet Borst(The Nether-lands Cancer Institute)for providing the MDCK II cells expressing MRP2and MDR1,and Dr.Yoshihiro Miwa(University of Tsukuba, Tsukuba,Japan)for providing pEB6CAGMCS/SRZeo vector.We also thank Dr.Makoto Sasaki for constructing Oatp1b2/Mrp2double-transfected cells and Chihiro Kondo for constructing human BCRP recombinant adenovirus.We also thank Sankyo Co.,Ltd.(Tokyo, Japan)for providing radiolabeled pravastatin,Novartis Pharma K.K.(Basel,Switzerland)for providing radiolabeled fluvastatin,As-traZeneca PLC(London,UK),for providing radiolabeled rosuvasta-tin,and Kowa Co.Ltd.(Tokyo,Japan)for providing radiolabeled pitavastatin and unlabeled statins.
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The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；
t h e-w a y-的用法
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。
way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句
t h e_w a y的用法大全
The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms. 从那天起,其他同学是夹着书本来上课,而他们却带着"失败"的思想负担来上课.
The way的用法及其含义（三） 三、the way的语义 1. the way=as（像） Please do it the way I’ve told you.请按照我告诉你的那样做。 I'm talking to you just the way I'd talk to a boy of my own.我和你说话就像和自己孩子说话一样。 Plant need water the way they need sun light. 植物需要水就像它们需要阳光一样。 2. the way=how（怎样，多么） No one can imagine the way he misses her.没人能够想象出他是多么想念她！ I want to find out the way a volcano has formed.我想弄清楚火山是怎样形成的。 He was filled with anger at the way he had been treated.他因遭受如此待遇而怒火满腔。That’s the way she speaks.她就是那样讲话的。 3. the way=according as （根据） The way you answer the questions, you must be an excellent student.从你回答问题来看，你一定是名优秀的学生。 The way most people look at you, you'd think a trash man was a monster.从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物。 The way I look at it, it’s not what you do that matters so much.依我看，重要的并不是你做什么。 I might have been his son the way he talked.根据他说话的样子，好像我是他的儿子一样。One would think these men owned the earth the way they behave.他们这样行动，人家竟会以为他们是地球的主人。
一．Way：“方式”、“方法” 1.表示用某种方法或按某种方式 Do it (in) your own way. Please do not talk (in) that way. 2.表示做某事的方式或方法 It’s the best way of studying [to study] English.。 There are different ways to do [of doing] it. 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句 正：I don’t like the way he spoke. I don’t like the way that he spoke. I don’t like the way in which he spoke.误：I don’t like the way how he spoke. 4. the way 的从句 That’s the way (=how) he spoke. I know where you are from by the way you pronounce my name. That was the way minority nationalities were treated in old China. Nobody else loves you the way(=as) I do. He did not do it the way his friend did. 二．固定搭配 1. In a/one way：In a way he was right. 2. In the way /get in one’s way I'm afraid your car is in the way， If you are not going to help，at least don't get in the way. You'll have to move－you're in my way. 3. in no way Theory can in no way be separated from practice. 4. On the way (to……) Let’s wait a few moments. He is on the way Spring is on the way. Radio forecasts said a sixth-grade wind was on the way. She has two children with another one on the way. 5. By the way By the way，do you know where Mary lives? 6. By way of Learn English by way of watching US TV series. 8. under way 1. Elbow one’s way He elbowed his way to the front of the queue. 2. shoulder one’s way 3. feel one‘s way 摸索着向前走；We couldn’t see anything in the cave, so we had to feel our way out 4. fight/force one’s way 突破。。。而前进The surrounded soldiers fought their way out. 5.. push/thrust one‘s way（在人群中）挤出一条路He pushed his way through the crowd. 6. wind one’s way 蜿蜒前进 7. lead the way 带路，领路；示范 8. lose one‘s way 迷失方向 9. clear the way 排除障碍，开路迷路 10. make one’s way 前进，行进The team slowly made their way through the jungle.
在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms.
“the way+从句”结构的意义及用法 首先让我们来看下面这个句子： Read the following passage and talk about it with your classmates. Try to tell what you think of Tom and of the way the children treated him. 在这个句子中，the way是先行词，后面是省略了关系副词that 或in which的定语从句。 下面我们将叙述“the way+从句”结构的用法。 1．the way之后，引导定语从句的关系词是that而不是how,因此，<<现代英语惯用法词典>>中所给出的下面两个句子是错误的：This is the way how it happened. This is the way how he always treats me. 2. 在正式语体中，that可被in which所代替；在非正式语体中，that则往往省略。由此我们得到the way后接定语从句时的三种模式：1) the way +that-从句2) the way +in which-从句3) the way +从句 例如：The way(in which ,that) these comrades look at problems is wrong.这些同志看问题的方法不对。
The way(that ,in which)you’re doing it is completely crazy.你这么个干法，简直发疯。 We admired him for the way in which he faces difficulties. Wallace and Darwin greed on the way in which different forms of life had begun.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 This is the way (that) he did it. I liked the way (that) she organized the meeting. 3.the way(that)有时可以与how(作“如何”解)通用。例如： That’s the way (that) she spoke. = That’s how she spoke. I should like to know the way/how you learned to master the fundamental technique within so short a time. 4．the way的其它用法：以上我们讲的都是用作先行词的the way，下面我们将叙述它的一些用法。
定冠词the的12种用法 定冠词the 的12 种用法，全知道?快来一起学习吧。下面就和大家分享，来欣赏一下吧。 定冠词the 的12 种用法，全知道? 定冠词the用在各种名词前面，目的是对这个名词做个记号，表示它的特指属性。所以在词汇表中，定冠词the 的词义是“这个，那个，这些，那些”，可见，the 即可以放在可数名词前，也可以修饰不可数名词，the 后面的名词可以是单数，也可以是复数。 定冠词的基本用法： (1) 表示对某人、某物进行特指，所谓的特指就是“不是别的，就是那个!”如： The girl with a red cap is Susan. 戴了个红帽子的女孩是苏珊。 (2) 一旦用到the，表示谈话的俩人都知道说的谁、说的啥。如：
The dog is sick. 狗狗病了。(双方都知道是哪一只狗) (3) 前面提到过的，后文又提到。如： There is a cat in the tree.Thecat is black. 树上有一只猫，猫是黑色的。 (4) 表示世界上唯一的事物。如： The Great Wall is a wonder.万里长城是个奇迹。(5) 方位名词前。如： thenorth of the Yangtze River 长江以北地区 (6) 在序数词和形容词最高级的前面。如： Who is the first?谁第一个? Sam is the tallest.山姆最高。 但是不能认为，最高级前必须加the，如： My best friend. 我最好的朋友。 (7) 在乐器前。如： play the flute 吹笛子
Way用法 A:I think you should phone Jenny and say sorry to her. B:_______. It was her fault. A. No way B. Not possible C. No chance D. Not at all 说明：正确答案是A. No way，意思是“别想！没门！决不！” 我认为你应该打电话给珍妮并向她道歉。 没门！这是她的错。 再看两个关于no way的例句： （1）Give up our tea break? NO way! 让我们放弃喝茶的休息时间？没门儿！ （2）No way will I go on working for that boss. 我决不再给那个老板干了。 way一词含义丰富，由它构成的短语用法也很灵活。为了便于同学们掌握和用好它，现结合实例将其用法归纳如下： 一、way的含义 1. 路线
He asked me the way to London. 他问我去伦敦的路。 We had to pick our way along the muddy track. 我们不得不在泥泞的小道上择路而行。 2. （沿某）方向 Look this way, please. 请往这边看。 Kindly step this way, ladies and gentlemen. 女士们、先生们，请这边走。 Look both ways before crossing the road. 过马路前向两边看一看。 Make sure that the sign is right way up. 一定要把符号的上下弄对。 3. 道、路、街，常用以构成复合词 a highway（公路），a waterway（水路），a railway（铁路），wayside（路边）
way/time的特殊用法 1、当先行词是way意思为”方式.方法”的时候,引导定语从句的关系词有下列3种形式： Way在从句中做宾语 The way that / which he explained to us is quite simple. Way在从句中做状语 The way t hat /in which he explained the sentence to us is quite simple. 2、当先行词是time时,若time表示次数时,应用关系代词that引导定语从句,that可以省略; 若time表示”一段时间”讲时,应用关系副词when或介词at/during + which引导定语从句 1.Is this factory _______ we visited last year? 2.Is this the factory-------we visited last year? A. where B in which C the one D which 3. This is the last time _________ I shall give you a lesson. A. when B that C which D in which 4.I don’t like the way ________ you laugh at her. A . that B on which C which D as 5.He didn’t understand the wa y ________ I worked out the problem. A which B in which C where D what 6.I could hardly remember how many times----I’ve failed. A that B which C in which D when 7.This is the second time--------the president has visited the country. A which B where C that D in which 8.This was at a time------there were no televisions, no computers or radios. A what B when C which D that